This invention relates to the application of genetic engineering techniques to plants. Specifically, the invention relates to compositions and methods for transformation of nucleic acid sequences into plant cell plastids.
Molecular biological techniques have enabled researchers to introduce pieces of DNA from one organism to another organism. Such techniques, referred to as recombinant DNA technology, have positively impacted the areas of medicine and agriculture. Conventional cloning methods have enabled the introduction of new pharmaceuticals and improved crops of agricultural importance. As the need for the introduction of multiple pieces of DNA and larger fragments of DNA into numerous target hosts increases, the need for novel cloning strategies increases accordingly.
The plastids of higher plants are an attractive target for genetic engineering. Plant plastids (chloroplasts, amyloplasts, elaioplasts, chromoplasts, etc.) are the major biosynthetic centers that in addition to photosynthesis are responsible for production of industrially important compounds such as amino acids, complex carbohydrates, fatty acids, and pigments. Plastids are derived from a common precursor known as a proplastid and thus the plastids present in a given plant species all have the same genetic content. Plant cells contain 500-10,000 copies of a small 120-160 kilobase circular genome, each molecule of which has a large (approximately 25 kb) inverted repeat. Thus, it is possible to engineer plant cells to contain up to 20,000 copies of a particular gene of interest, which potentially can result in very high levels of foreign gene expression.
Plastid transformation has been restricted to a few dicot species. In all successful experiments (Svab et al., Proc. Natl. Acad. Sci. USA 87, 8526-8530, 1990; Svab and Maliga, Proc. Natl. Acad. Sci. USA 90:913-917, 1993; Sikdar et al., Plant Cell Reports 18:20-24, 1998; Sidorov et al., Plant Journal 19(2):209-216, 1999), green leaves that contain developed chloroplasts were the target material used for particle bombardment. The most efficient selectable marker for isolation of plastid transformants is aadA, which confers resistance to the antibiotics spectinomycin and streptomycin (Svab et al., Proc. Natl. Acad. Sci. USA 87, 8526-8530, 1990; Svab and Maliga, Proc. Natl. Acad. Sci. USA 90:913-917, 1993; Sikdar et al., Plant Cell Reports 18:20-24, 1998; Sidorov et al., Plant Journal 19(2):209-216, 1999). NPTII, conferring resistance to kanamycin, has been reported for tobacco (Carrer et al., Mol Gen Genet 241:49-56, 1993) and glyphosate (U.S. Patent Application 200242934) were also used for selecting plastid transformants.
Recently, plastid transformation in rice was reported using a nongreen embryogenic culture as initial material for transformation (Khan and Maliga, Nature Biotechnology 17(9):910-915, 1999). Selection was for streptomycin resistance conferred by aadA, as rice is not sensitive to spectinomycin. Selection was applied for only two weeks in liquid suspension and during the subsequent plant regeneration phase. Regenerated plants were chimeric, with some leaves containing sectors with transformed plastids. However, the transformed cells were also apparently only heteroplasmic, having both transformed and non-transformed plastids. This pattern of heteroplasmy is most likely due to the short period of selection that did not allow enough time for amplification of transformed plastids prior to plant regeneration.
However, a need still exists for a method of producing homoplasmic monocotyledonous plants, which are most useful. The present invention should be applicable to all monocots.